Method of using antiviral composition

ABSTRACT

A composition having antiviral and antibacterial effects is disclosed which comprises the dimeric forms of enzymes selected from lysozyme and ribonuclease and a pharmaceutically acceptable carrier. These dimeric forms are more effective in treating a variety of human and animal diseases because they are much less cytotoxic than the monomeric forms of the enzyme. A method for the use of these compositions is also disclosed which comprises applying an effective amount of the composition to the infected area.

This is a division of application Ser. No. 07/865,002, filed Apr. 8,1992 now U.S. Pat. No. 5,466,449, which is a division of applicationSer. No. 07/459,738, filed Jan. 26, 1990, now U.S. Pat. No. 5,200,182.

FIELD OF THE INVENTION

The present invention relates to novel antiviral and antibacterialcompositions comprising polymerized enzymes along with apharmaceutically acceptable carrier, and a method for their use.

BACKGROUND OF THE INVENTION

The ever growing number of bacterial strains and viral diseases whichare resistant to antibiotics have made it necessary to introduce newkinds of drugs in order to treat humans and animals. Among the manypresent treatments and medicines, it has been known to use enzymes inmonomeric form in order to provide therapeutic effects in patientsafflicted with various diseases. Enzymes are catalytically activeproteins which perform almost all major life processes in organisms.Thus, many enzymes, either individually or in certain combinations, havebeen isolated for their physiocochemical, physiological, or biologicaleffects.

Among the various enzymes or which certain therapeutic effects have beendocumented are lysozyme and ribonuclease. Lysozyme has been known since1922, the year when it was discovered by Fleming. Only after 1950,however, were the enzymatic functions of lysozyme revealed. Since thistime, the compound has been a subject of intense physiocochemical,physiologic and clinical research, but the extent of this compound'sbiological significance has still yet to be determined. Thus far,lysozyme has been observed to have various therapeutic properties, suchas antiviral, antibacterial, anti-inflammatory and antihistaminicproperties. The antibacterial effect appears to be based on thehydrolysis of the beta-1-4-glycoside bond between N-acetylomuraminicacid and N-acetyloglucosamine, both contained in the bacterial wall.

The presence of lysozyme in phagocytotic cells is also well documented.Research in this area has shown that the intracellular lysozymecontained in lysozymes is responsible for digesting the phagocytizedbacteria. In humans, lysozyme has been observed to stimulatephagocytosis at a physiological concentration of 10-400 mg/ml.

Other properties of lysozyme have also been documented. For instance, itappears that lysozyme reduces body temperature during the infectionprocess, where temperature is a response to endogenic pyrogens liberatedby toxins. It also appears that lysozyme participates in immunologicalprocesses by stimulating the synthesis of gamma globulins, opsonins, andother antibodies. Still further, it has been suggested that lysozyme hasa strong anti-inflammatory effect. Despite these known beneficialproperties of lysozyme, despite numerous research projects and theproduction of pharmaceutical preparations based on lysozyme, the use ofthis enzyme for therapeutical purposes has been vastly limited.

Another group of enzymes which have been studied for their variousbiological effects are the ribonucleases. These are a group of enzymescommonly found in many animal and plant organisms as well as inbacterial cells. The study of their properties and research into methodsof isolation was initiated in 1955 by Schmidt and McDonald. Among thefindings based on this enzyme, it was found that in cancerous tissuesthe activity of ribonucleases was considerable reduced. For example, itwas discovered that leukemogenic viruses drastically diminished theactivity of acid ribonuclease in mice. Also, in mice with viralleukemia, a considerable decrease in acid ribonuclease activity wasfound in mitochondria and microsome fractions obtained from the spleentissue of those animals.

The studies cited above suggest that the decrease in activity ofribonuclease is somehow closely connected with the infections caused bythe virus. It has thus been suggested that the ribonuclease enzymes maypossess some antiviral activity. Again, however, at present, there havebeen no known reports on the preparation of compounds employing thisenzyme as an antiviral agent.

One of the main reasons why such potentially beneficial enzymes have notas yet been widely used for their therapeutic effects is the observedcytotoxic effect of the monomeric forms of these and other enzymes. Intests with cultured fibroblasts, there has been an observed cytotoxiceffect from both the lysozyme and ribonuclease monomers at even verysmall quantities. Clearly, the potential beneficial effects from theseand other enzymes could be achieved if an effective way of controllingthese cytotoxic effects could be developed. What is desired, therefore,is to develop compositions based on lysozyme, ribonuclease, or othersimilar enzymes, which can effectively be used to treat viral orbacterial disease or other harmful conditions without the cytotoxiceffects normally observed when the enzymes are used in the monomericform.

SUMMARY OF THE INVENTION

In accordance with the present invention, it has been discovered that anantiviral or antibacterial composition having as its active ingredientlysozyme or ribonuclease, yet which does not exhibit cytotoxic effectscan be prepared by using the dimeric forms of these enzymes. Bypreparing compositions using as the active ingredient a lysozyme orribonuclease dimer and a pharmaceutically acceptable carrier, a numberof infectious diseases can be successfully treated without appreciablecytotoxic effects.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The antiviral and antibacterial compositions of the present inventioncan be prepared by first obtaining lysozyme and ribonuclease in theirmonomeric form. The lysozyme monomers (Catalog No. 28260) andribonuclease monomers (Catalog No. 34388) used in the preparation ofcompounds prepared in accordance with the present invention, wereobtained from Serva Feine Biochemica, Gmbh und Cooperation, D69-000,Heidelberg. These enzyme monomers can be polymerized into dimers by anyconventional method currently used in the art. However, particularlypreferred, is the enzyme polymerization carried out and disclosed inCarlsson et al, Biochemistry Journal, 173: 723-737 (1978). Othermethods, such as that described in Sorrentino et al., Eur. J. Biochem.124: 183-9 (1982), can also be utilized. It has also been observed thata particularly useful ribonuclease dimer which can be employed in thecompositions of the present invention is a dimer prepared frompancreatic ribonuclease A, isolated from animal pancreatic tissues.

It has been observed that compositions containing as the activeingredient a lysozyme or ribonuclease dimer and a pharmaceuticallyacceptable carrier are effective in treating a variety of bacterial andviral diseases without undesirable cytotoxic effects. These potentiallyharmful effects of the enzymes were tested in comparative studies usingboth the monomeric and dimeric forms of lysozyme and ribonuclease. Inthese studies, various concentrations of monomers and dimers of lysozymeand ribonuclease were administered to cultures of green monkey kidney(GMK) fibroblasts. It was observed that the lysozyme monomer proved tobe cytotoxic to fibroblasts after 24 hours at concentrations of 0.1mg/ml and 1.0 mg/ml. After three days of incubation, a cytotoxic effecton fibroblasts was observed even at a concentration of 0.01 mg/ml, whichaffected 50% of the incubated cells. After five days, 75% of thecultivated cells were affected by the cytotoxic activity of the lysozymemonomer at concentrations of 1.0 and 0.1 mg/ml. Comparatively, thelysozyme dimer showed no cytotoxic effect in any concentration used inthese tests, not even after a seven day period. These studies showedthat the dimeric form of lysozyme was approximately 100 times less toxicto GMK fibroblasts than the monomeric form.

The study with regard to ribonuclease showed a similar lack of cytotoxiceffect for the dimeric form. In the study, ribonuclease proved to becytotoxic to GMK fibroblasts in a five day culture for concentrations aslow as 0.0001 mg/ml. After seven days of culture, cytotoxic effects ofthe ribonuclease monomers at concentrations of 0.01 mg/ml and aboveeliminated 100% of the cultivated cells. As a contrast, the ribonucleasedimer showed no cytotoxic effects whatsoever on the GMK fibroblasts atall concentration levels, not even after a seven-day incubation. Thedimeric form of ribonuclease therefore, was observed to be approximately1,000-10,000 times less toxic to the fibroblasts than the monomericform. These tests clearly showed that the cytotoxic effects whichnormally accompany use of the monomeric forms of enzymes such aslysozyme and ribonuclease could be virtually eliminated if those enzymeswere utilized in their dimeric forms.

Further research has shown that despite the lack of appreciablecytotoxic effects, lysozyme and ribonuclease in their dimeric form canbe extremely effective in treating viral and bacterial infections. Intests involving fertilized hen eggs and a Sendai virus strain, lysozymedimer was intraamniotically injected into eggs at variousconcentrations. Into each egg was also injected two units of the Sendaivirus. After incubation, the amniotic and allantoic fluids werecollected from infected and control eggs and compared. These testsindicated that the lysozyme dimer was able to inhibit the replication ofthe Sendai virus cultivated in ten day old fertilized hen eggs, even ata concentration as low as 0.01 mg/ml. Similar tests using lysozyme andribonuclease dimers have shown a bacteriostatic effect of these dimericenzymes on strains of Streptococcus bacteria.

Compositions prepared from lysozyme or ribonuclease dimers in accordancewith the present invention, therefore, can be used to treat a variety ofviral and bacterial infections. The compositions can be prepared in avariety of forms, and administration of these enzyme-carriercompositions can be carried out internally or externally for aparticular human or animal patient depending on the disease that needsto be treated. For internal diseases, such as ear infection, mastitis,stomach or vaginal tract infections, the dimeric compositions of theinvention can be suitably prepared and administered orally,intravenously, parenterally, via a suppository, or any other methodwhich will enable the dimeric solution to reach the infected area. Forexternal conditions, such as viral or bacterial skin disease, infectedwounds, or herpes or other sexual diseases with external effects, thecompositions of the invention can be administered topically to thepatient in any of a variety of suitable forms.

The particular nature of the diseases or infection treated, therefore,will determine the proper form for the composition of the presentinvention. For external treatments, the composition can be administeredin such various forms as ointments, lotions, solutions, oils, etc. Wherean internal application is necessary, a number of suitable forms such asdrops, tablets, solutions, capsules, dentrifices, etc. can be employed.The particular form of the composition as applied to the patient willalso thus determine the nature of the pharmaceutically acceptablecarrier used in the dimeric enzyme composition. Among the many suitablecarriers which can be used are hydrophilic bases, physiologicallyacceptable salt solutions, water, ointments, powders, etc.

Enzymatic treatment of viral or bacterial infections without cytotoxiceffects is thus provided in the present invention by administering to ahuman or animal patient an effective amount of the dimeric compositionsdiscussed above. By effective amount is meant that amount which isnecessary to produce antiviral or antibacterial effects. The amountneeded to effect treatment will vary in each case depending on thenature of the disease treated and the form of the dimeric compositionprovided. In general, the composition of the present invention isadministered at a dosage level of from 0.1 to 5.0 mg/kg of body weight,with a range of 1.0 to 2.0 mg/kg/body weight particularly preferred. Inthe case of topical treatment, ointments prepared using 4-20 mg of thedimer in approximately 200 ml of a solution of water, paraffin andpropylene glycol has been effective if applied 4-5 times per day.Dosages in these ranges should be sufficient to treat a number of viralor bacterial diseases without the harmful cytotoxic effects that wouldaccompany treatments with enzyme monomers.

The following examples are presented as illustrative only of the presentinvention and are not intended to limit its scope in any way:

EXAMPLE 1

Comparative research was carried out concerning the cytotoxic effect ofmonomers and dimers of lysozyme and pancreatic ribonuclease A on aculture of green monkey kidney (GMK) fibroblasts. These tests werecarried out by applying monomers and dimers to the fibroblast culture atconcentrations of 0.0001 to 1.0 mg/ml. The cultures were then incubatedfor seven days, after which the cultures were examined for cytotoxicity.The results of these tests are presented in Tables 1 and 2.

As can be observed from Table 1, the lysozyme monomer proved to becytotoxic to fibroblasts after 24 hours at concentrations of 0.1 mg/mland 1.0 mg/ml. After three days of incubation, the monomer showedcytotoxic effects on fibroblasts even at a concentration of 0.01 mg/ml,affecting 50% of the incubated cells. After five days, 75% of thecultivated cells were affected by the cytotoxic activity of the lysozymemonomer at concentrations of 1.0 mg/ml and 0.1 mg/ml.

In contrast, the dimeric form of lysozyme showed no cytotoxic effect inany concentration used in the test even after completion of the sevenday incubation. The studies thus showed that the lysozyme dimer provedto be about 100 times less toxic to GMK fibroblasts than the lysozymemonomer.

As can be observed in Table 2, the monomer of pancreatic ribonuclease Aproved to be cytotoxic to GMK fibroblasts in the five day culture evenat concentrations as low as 0.0001 mg/ml. After seven days of culturing,the cytotoxic effect of pancreatic ribonuclease A at concentrations of0.01 mg/ml and above was of sufficient strength to eliminate 100% of thecultivated cells.

As was similar to the case with the lysozyme dimer, the dimeric form ofpancreatic ribonuclease A showed no cytotoxic effect on GMK fibroblastsat any concentration used in the test for the duration of the seven dayperiod. The results thus indicated that the dimer of pancreaticribonuclease A was about 1,000 to 10,000 times less toxic to GMKfibroblasts than the pancreatic ribonuclease A monomer.

                  TABLE 1                                                         ______________________________________                                        Tests for cytotoxicity of MONOMER of lysozyme                                 and DIMER of lysozyme                                                         Type of Conc.     After  After   After After                                  Preparation                                                                            mg/ml     24 hr   3 days                                                                               5 days                                                                             7 days                                 ______________________________________                                        Control                  0       0     0                                      Lysozyme                                                                                       1.0                                                                                  2                                                                                   2        3                                                                                  3                                 MONOMER                 10.1                                                                                2        3                                                                                  3                                                         0.01                                                                                2        2                                                                                  2                                         0.001         0       0        0                                                                                  0                                         0.0001       0        0        0                                                                                  0                                 Lysozyme                                                                                       1.0                                                                                  0                                                                                   0        0                                                                                  0                                 DIMER                   0                                                                                   0        0                                                                                  0                                                        0      0        0                                                                                  0                                                       0  0.001                                                                              0        0                                                                                  0                                         0.0001       0        0        0                                                                                  0                                 ______________________________________                                         The tests were performed 3x on a culture of GMK/green monkey                  kidney/fibroblasts.                                                           0  cytotoxicity = 0%                                                          1  cytotoxicity = 25%                                                         2  cytotoxicity = 50%                                                         3  cytotoxicity = 75%                                                         4  cytotoxicity = 100%                                                   

                  TABLE 2                                                         ______________________________________                                        Tests for cytotoxicity of pancreatic ribonuclease A                           MONOMER and pancreatic ribonuclease A DIMER                                   Type of Conc.     After  After   After After                                  Preparation                                                                           mg/ml      24 hr   3 days                                                                              5 days                                                                              7 days                                 ______________________________________                                        Control           0      0       0     0                                      Pancreatic                                                                             1.0             1                                                                                 2        4                                                                                   4                                 ribonuclease                                                                          0.1             0                                                                                   1       3                                                                                   4                                 A MONO-   0.01         0      0       3                                                                                   4                                 MER                   00.001                                                                                0       2                                                                                   3                                         0.0001       0        1       1                                                                                   2                                 Pancreatic                                                                             1.0            0                                                                                   0       0                                                                                   0                                 ribonuclease                                                                          0.1             0                                                                                   0       0                                                                                   0                                 A DIMER     0.01       0      0       0                                                                                   0                                         0.001         0       0       0                                                                                   0                                 ______________________________________                                         The test was performed 3x on a culture of GMK/green monkey                    kidney/fibroblasts.                                                           0  cytotoxicity = 0%                                                          1  cytotoxicity = 25%                                                         2  cytotoxicity = 50%                                                         3  cytotoxicity = 75%                                                         4  cytotoxicity = 100%                                                   

EXAMPLE 2

The dimer of lysozyme was studied with regard to its antiviral effects.In the experiments, lysozyme dimer was injected into ten day-oldfertilized hen eggs in concentrations of 10.0 mg/ml, 1.0 mg/ml, 0.1mg/ml, 0.01 mg/ml and 0.001 mg/ml. A Sendai virus strain(hemagglutinational titre=1:128 HA) was added to each concentration ofthe dimer in an amount of two hemagglutinational units. Afteradministration of the lysozyme dimer and the virus, the eggs wereincubated for 72 hours at 37° C. After the incubation period, theamniotic and allantoic liquids were collected from the infected eggs andsubject to a hemagglutination test by means of micromethods with the useof a Takatsy set and hen blood corpuscles. The experiments were thenrepeated. The results obtained in these experimental trials arepresented in Table 3. As can be observed from Table 3, the lysozymedimer inhibited the replication of Sendai virus cultivated in tenday-old fertilized hen eggs, even at concentrations as low as 0.01mg/ml.

TABLE 3

The effect of lysozyme DIMER on Sendai virus strain.

                  TABLE 3                                                         ______________________________________                                        The effect of lysozyme DIMER on Sendai virus strain.                          Concen-                                                                       tration of    Hemagglutination inhibition/                                    lysozyme        hemagglutination test on hen red blood                        DIMER              cells according to Takatsy method                          in mg/ml    September 1987                                                                            November 1987                                         ______________________________________                                        10.0        +++         not investigated                                      1.0         +++         +++                                                   0.1         +++         +++                                                   0.01        +++         +++                                                   0.001       ++          no inhibition                                         ______________________________________                                         +++ full inhibition, 1:256                                                    ++ limited inhibition, 1:32                                              

1. Tests were performed on Sendai virus strain. The viralhemagglutination titre was 1:128 HA.

2. 10 day-old, fertilized hen eggs served as an experimental model.

3. An identical quantity of Sendai virus--2 hemagglutinationalunits--was added to each concentration of lysozyme DIMER. At the sametime a control test for lysozyme DIMER was initiated in order to findout if it has hemagglutinational properties--the result was negative.Consecutive concentrations of lysozyme DIMER plus 22 HA units of thevirus were then used to infect intraamniotically 4 hen eggs. The eggswere incubated for 72 hours at 37° C.

4. After the incubation period, the amniotic and allantoic fluids werecollected from the infected eggs. A hemagglutinational test wasperformed (hemagglutinational micromethod) with the use of Takatsy testset and hen red blood cells.

EXAMPLE 3

The effects of the dimeric forms of lysozyme and pancreatic ribonucleaseA on bacteria were tested on several pathogenic strains collected fromcows with mastitis. The effect of different concentrations of lysozymedimer on three strains (Streptococcus agalactiae, S. dysgalactiae and S.liberis) are presented in Table 4. These test results showed that allthree of the Streptococcus strains proved to be sensitive to theactivity of the lysozyme dimer. This was most evident in the case of S.liberis which was affected by the activity of the dimer at aconcentration as low as 1.25 mg/ml. Bacteriostatic effects on the otherstrains of Streptococcus were observed at concentrations starting atabout 10 mg/ml.

In Table 5, the effects of pancreatic ribonuclease A dimer and lysozymedimers on pathogenic bacteria strains cultivated from human patients ispresented. As can be observed from the table, pancreatic ribonuclease Adimer was most effective on bacterial strains of Pseudomonas aeruginosa,Escherichia coli and Proteus vulgarus, particularly at a concentrationranging of from about 5 to 10 mg/ml. Bacterial strains of Staphylococcusand Streptococcus were found to be sensitive to the lysozyme dimer, alsoat concentrations of about 5 to 10 mg/ml. Sensitivity tests wereperformed in accordance with generally accepted international principlesrecommended by the WHO.

EXAMPLE 4

The effect of the lysozyme dimer and the pancreatic ribonuclease A dimeron the proliferation of K-562 Erythroleukemic cell lines was determinedby

TABLE 4

MIC--Minimal Inhibitory Concentration of lysozyme DIMER INmg/ml--Bacterial strains were cultivated on samples collected from cowswith mastitis

                  TABLE 4                                                         ______________________________________                                        MIC - Minimal Inhibitory Concentration of lysozyme                            DIMER IN mg/ml - Bacterial strains were cultivated                            on samples collected from cows with mastitis                                              Lysozyme DIMER in mg/ml                                           Bacterial Strains                                                                           1.25     2.5   5.0   10.0 20.0                                  ______________________________________                                        Streptococcus agalactiae                                                                    +        +     +     -    -                                     Streptococcus dysgalactiae                                                                    +          +    +     -    -                                  Streptococcus liberis                                                                              -    -    -     -     -                                  ______________________________________                                         - = no proliferation of bacteria                                              + = proliferation of bacteria                                            

Sensitivity tests were performed in accordance with generally acceptedinternational principles recommended by the WHO.

TABLE 5

MIC--Minimal Inhibitory Concentration of pancreatic ribonuclease A DIMERin mg/ml Bacterial strains were cultivated on samples collected frompatients.

                  TABLE 5                                                         ______________________________________                                        MIC - Minimal Inhibitory Concentration of                                     pancreatic ribonuclease A DIMER in mg/ml                                      Bacterial strains were cultivated on                                          samples collected from patients.                                                        Pancreatic                                                                         ribonuclease                                                                                    Lysozyme DIMER                               Bacterial    A DIMER in mg/ml                                                                              in mg/ml                                         strains   2.5    5.0    10.1 20.0 2.5  5.0 10.0 20.0                          ______________________________________                                        Pseudomonas                                                                             +      -      -    -    +    +   +    +                             aeruginosa                                                                    Escherichia                                                                             +         +      -   -     +    +                                                                                +     +                          coli                                                                          Proteus   +         +      -   -     +    +                                                                                +     +                          vulgaris                                                                      Staph.aureus/                                                                           +         +      +    +     +                                                                                 -                                                                               -     -                           Standard strain                                                               209/                                                                          Staph.aureus/                                                                                  + +    +    +    +    -   -    -                             pathogenic strain                                                             from patients                                                                 Staph.aureus                                                                             +     +      +    +    +    +   -    -                             MRSA strain                                                                   nr 11704                                                                      Staph.aureus                                                                               +      +      +    +     +                                                                                 +                                                                                -    -                           MRSA strain                                                                   nr 11708                                                                      Strept.pyogenes                                                                         +         +      +    +     +                                                                                 -                                                                               -     -                           pathogenic strain                                                             from patient                                                                  ______________________________________                                         - = no proliferation of bacteria                                              + = proliferation of bacteria                                                 MSRA  Methicilin Resistent Staphylococcus aureus. Strains collected from      patients. Sensitivity tests were performed in accordance with generally       accepted international principles recommended by the WHO.                

TABLE 6

The effect of lysozyme DIMER on the proliferation of K-562erythroleukemic cell lines. Cells of K-562 line were used at aconcentration of 10⁵ ml. The effect was estimated after 24 hours ofculture at 37° C. and with a 5% CO₂ flow.

                  TABLE 6                                                         ______________________________________                                        The effect of lysozyme DIMER on the proliferation of                          K-562 erythroleukemic cell lines. Cells of K-562                              line were used at a concentration of 10.sup.5 ml.                             The effect was estimated after 24 hours of                                    culture at 37° C. and with a 5% CO.sub.2 flow.                                         Proliferation of K-562 cells in                               Concentration         24 hours in vitro culture                               lysozyme DIMER  Number of                                                                              Percentage of                                        in mg/ml                     Dead Cells                                       ______________________________________                                        Control         190,000  2-3%                                                 1.0                             100%s of                                                      cells                                                         0.1                                95%0                                       0.05                                99%                                       ______________________________________                                    

TABLE 7

The effect of pancreatic ribonuclease A DIMER on the proliferation ofK-562 erythroleucemic cell lines. Cells of K-562 line were used at aconcentration of 10⁵ ml. The effect was estimated after 24 hours ofculture at 37° C. and with a 5% CO₂ flow.

                  TABLE 7                                                         ______________________________________                                        The effect of pancreatic ribonuclease A DIMER on                              the proliferation of K-562 erythroleucemic cell                               lines. Cells of K-562 line were used at a                                     concentration of 10.sup.5 ml. The effect was                                  estimated after 24 hours of culture at                                        37° C. and with a 5% CO.sub.2 flow.                                    Concentration of                                                                              Proliferation of K-562 cells in                               pancreatic            24 hours in vitro culture                               ribonuclease    Number of                                                                              Percentage                                           A DIMER                           of Dead Cells                               ______________________________________                                        Control         207,000  2-5                                                  1.0                                   740                                     0.1                                   14                                      0.05                                    7                                     ______________________________________                                    

applying various concentrations of the dimers to cells from these lines.The results of these tests are presented in Tables 6 and 7. In short,all concentrations of the lysozyme dimer used in the experiment showedstrong cytopathogenic effects on the K-562 cells. Additionally, as canbe observed in Table 7, the pancreatic ribonuclease A dimer also had asimilar effect on the Erythroleukemic cell line, but only atconcentrations of 1.0 mg/ml.

EXAMPLE 5

The effect of lysozyme dimer on purulent otitis media in dogs wasexamined. The study was undertaken using 19 dogs of different races withvarious forms of the disease. The disease was characterized by aninflammatory process which on average lasted for about 7-14 days, but inone case lasted for nine months. In seven of the dogs, the purulentdischarge from the inflamed ear was examined for an identification ofbacterial strains therein before treatment was undertaken. Theseculturing tests showed the presence of Staphlococcus germs, bluepusbacillis, Pseudomonas aeruginosa, Coccidia species, and variousbacilli species.

The afflicted dogs manifested various signs that they were in pain, suchas shaking their heads and trying to reach the infected ear with theirpaws. The dogs generally had impaired appetites and heightenedtemperatures (39.2-41.2° C.). Eighteen of the dogs in the study had notbeen given any previous treatment with any pharmacological means. Onedog, in which the infection had persisted for nine months, had beengiven several antibiotics, but these had not been effective in treatingthe condition.

The dogs were treated with a composition of the present invention whichconsisted of a solution of 20 mg of lysozyme dimer and 25 ml ofphysiological salt. The composition was applied in the form of drops,and ten drops were placed into the inflamed ear four-five times a day.After the first day of treatment, a marked improvement was alreadyobserved: temperatures dropped, the dogs apparently felt morecomfortable, and had better appetites. The symptoms of purulentinflammation receded fully between the third and sixth day of thetreatment. In the dog which had been previously treated with antibioticsunsuccessfully for nine months, successful recession of the disease wasachieved after ten days. Lysozyme dimer was thus shown to be effectivein treating purulent otitis media in dogs.

EXAMPLE 6

The effects of lysozyme dimer on cows with mastitis was examined. Sixcows with mastitis were used in the study. These cows showed signs ofthe disease such as temperatures above 40.5° C. and an impairedappetite. In all six cases, the treatment began on the second day of thedisease. Before the compositions were administered, samples of milk werecollected for bacterial examination. The cultures were observed tocontain microbes such as Staphlococcus and Streptococcus agalactiae.

Lysozyme dimer was given to each cow through syringe injection in theinfected teats at a dosage of 40 mg in a solution of 50 ml ofphysiological salt twice a day. It was found that after only 24 hours,the body temperature was normal again, and the appetite returned. Afterthree days, all cows under treatment showed no symptoms of the disease.The treatment was thus only continued up to four days, after whichexamination of the milk showed that the pathogenic microbes found beforetreatment had disappeared. Further, no changes were found in the milkthat would indicate a subclinical mastitis. In none of the treated cowswas there found a decrease in milk yield, nor was the patency of teatsimpaired. After 24 hours of the lysozyme dimer treatment, no blockingsubstances were found in the milk of the treated cows. The quickdisappearance of disease symptoms, as well as full retention of milkingcapacity thus indicates that the lysozyme dimer compositions of thepresent invention may be used successfully in the treatment of bovinemastitis. This treatment will be of particular economic importance forpreventing mastitic infections caused by Staphlococcus and Streptococcusbacterial strains, which currently cost the dairy industry approximately$5.4 billion a year.

EXAMPLE 7

Canine parvovirus (CPV) infection was treated by oral administration oflysozyme dimer. In twenty-seven dogs of different races and weight, agedfrom three months to six years, veterinary surgeons found a symptomcomplex typical for parvovirus infection. All animals in the tests hadhigh temperatures (40-41.6° C.), frequent paroxysms of profuse vomiting,numerous and characteristically fetid diarrhea stools, as well assymptoms of dehydration and apathy. The animals also gave impressions ofintense suffering. Treatment began on the average between the third andfifth day of infection, depending on how soon an animal's owner broughtthe pet to the veterinary station. The infected dogs were give lysozymedimer at a dose of 1-2 mg per kg of body weight two times a day. Toanimals which were still able to drink, the lysozyme dimer wasadministered in drinking water. Animals unable to drink were given thepreparation through a probe in a solution of physiological salt.

Of the twenty-seven dogs under treatment, twenty-five dogs regained fullphysical fitness after 3-5 days of treatment. Typically, even during thefirst day, there was observed a marked decrease in the number of stoolsand attacks of vomiting, and in the majority of the dogs these symptomsreceded totally after two days of treatment. In a few dogs, thesesymptoms disappeared even after the first dose of the preparation. Noside effects connected with the administration of the lysozyme dimercomposition were observed in any animal. These clinical tests show thatthe lysozyme dimer composition of the invention may be successfully usedon dogs with parvovirus infection.

EXAMPLE 8

The effects of the lysozyme dimer composition of the present inventionon certain dermatological diseases in humans were examined. Tests werecarried out with several dozen human patients aged from 15 to 35 andsuffering from various skin diseases which had been previously butunsuccessfully treated with conventional methods. The following diseaseswere identified in this group:

1 Forunculosis chronica--2 cases

2 Sycosis barbae--1 case

3 Impetigo contagiosa--11 cases

4 Acne vulgaris--22 cases

5 Resacea--6 cases

6 Varicose ulcer--12 cases

In some of the patients from this group, the treatment was preceded bybacteriological culture tests. In most cases, Staphlococcus aureus wascultivated from the collected material. The treatment consisted inadministering four times a day an ointment containing 20 mg of lysozymedimer. A particularly preferred formula for the ointment is as follows:

    ______________________________________                                        Lysozyme dimer         20.0   mg                                              Acetylstearoyloxy alcohol                                                                                  25.0                                                                           mg                                              Paraffin liquidum             mg     10.0                                     Span 60                       mg                5.0                           Tween 60                      mg               8.0                            Propylene glycol              mg      10.0                                    Aseptina M                    mg             0.3                              Aseptina P                    mg             0.16                             Distilled water               ml      200.0                                   ______________________________________                                    

In all patients, the various skin conditions disappeared within 10-12days, and in some cases clearing was observed after 3 days. In patientswith chronic Forunculosis, the treatments lasted up to 4-5 weeks, andthose with varicose ulcer generally took from 2-12 weeks to recoverdepending on how severe the skin condition was, and how long the diseasehad developed before the treatment. The results obtained in this studysuggests that lysozyme dimer may be used successfully for a variety ofskin diseases.

EXAMPLE 9

Various infectious diseases of the genital region were treated using alysozyme dimer composition of the present invention. Nine women, agedfrom 25-49 were treated in these tests, seven patients having Colpitischronica, one patient having Douglas' abscess, and one withBartholinitis. The patients with Colpitis chrorica were givenintra-vaginal suppositories containing 10 mg of lysozyme dimer in 2 ccmof hydrophilic base. The suppositories were administered two times aday, for a period of 7 days. In all patients, there was observed a totalrecession of the inflammation of the genital area. In addition,leucorrhea and other symptoms disappeared as well. The patient withDouglas' abscess was given 20 mg of lysozyme dimer in 5 ml of 0.9% NaClsolution two times a day over a span of 4 days. The solution was applieddirectly into the Douglas' cavity. Before each administration oflysozyme dimer, purulent content was aspired from the Douglas' cavity.These cultures indicated the presence of Streptococcus haemolyticus andbacterium coli. The patient's abnormally high body temperature wasreduced to normal values within 24 hours after the first administrationof lysozyme dimer. Pain symptoms as well receded during this time. Onthe fourth day after the second administration of the lysozyme dimercomposition, no pus was found in Douglas' cavity. In this case, therewas a reoccurrence of the disease after three weeks, but another twodoses of lysozyme dimer administered into the Douglas' cavity was ableto bring the disease process under control.

The patient with Bartholinitis was treated with one dose of 20 mg oflysozyme dimer in 1 ccm of 0.9% NaCl, which was administered immediatelyinto the suppurated gland, after the aspiration of purulent content fromit. After 4 days, this patient was found to be completely cured. During4 months of subsequent observations, there were no reoccurrences of thisdisease. These clinical tests indicate that lysozyme dimer has anextremely beneficial therapeutic effect in cases of certain infectiousdiseases of the genital region in women. Additionally, it appears thatit will be possible to treat local abscess by administering lysozymedimer immediately into the cavities with purulent content.

EXAMPLE 10

Effect of a lysozyme dimer solution on infected wounds was studied. Inthis group, there were 4 patients with infected post-operative wounds; 2women after laparotomy, one woman after toe amputation due to nucrosisin the course of diabetic angiopathy, and one man after amputation oflower extremity due to Burger's disease. In all cases, moistapplications and lavations with a solution of lysozyme dimer in 5 ml of0.9% NaCl were given four times a day. In the patients with suppuratingwounds after laparotomy, complete cure was effected after 4 and 6 dayperiods. In the other patients, cure was effected after 21 days and 5months, respectfully. These tests indicate that lysozyme dimer may beused as a therapeutic means without side effects in the treatment ofinfected post-operative wounds.

EXAMPLE 11

Clinical observations were made on patients with Herpes genitalistreated with a ribonuclease A dimer composition. The study groupincluded five patients who were women aged from 23-36 years old. Four ofthem had developed the disease for the first time, while one of thewomen had the disease for the third time. All patients were in theperiod of blistering, which is normally between the third and fifth dayof the disease, and complained of very strong pain in the perinealregion, particularly during urination. In all patients, we foundswelling and infection of labia as well as numerous blisters filled withturbid fluid on mucous labial membranes and on the external skin of thethigh and anal regions. In all patients the inguinal lymphatic noduleswere enlarged and painful.

The treatment involved application of an ointment containing the dimerof ribonuclease A four to five times daily on blisters and infectedareas of mucous membrane. The ointment applied had the followingformula:

    ______________________________________                                        Ribonuclease dimer     20.0   mg                                              Acetystearoyloxy alcohol                                                                                 25.0                                                                             mg                                              Paraffin liquidum             mg  10.0                                        Span 60                       mg             5.0                              Tween 60                      mg             8.0                              Propylene glycol              mg   10.0                                       Aseptina M                    mg          0.3                                 Aseptina P                    mg          0.16                                Distilled water               ml   200.0                                      ______________________________________                                    

The patients receiving the dimer treatment reported that after severalminutes and at least within one hour since the first application of theointment, pain diminished substantially, and disappeared totally overthe next 10-20 hours. Physical examination showed that in four patients,pathological changes receded totally after three days of treatment. Inthe other patient, pathological conditions were totally eliminated afterfive days. In none of these patients did new blisters emerge after theapplication of the ointment containing pancreatic ribonuclease A dimer.The above study shows that ointments containing dimers of the presentinvention can be used as a successful treatment for Herpes genitalis.

EXAMPLE 12

More than 100 patients of various ages were treated for labial herpesusing compositions of the present invention. In all of these patients,symptoms included swelling of the upper lip region, reddening andnumerous blisters filled with turbid fluid. All patients complained ofpain in the skin in areas affected by the disease, and in addition therewas a sensation of tension in the tissues. Treatment involvedapplication locally, for four to five times daily, of an ointmentcontaining pancreatic ribonuclease A dimer.

All of the patients treated without exception stated that pain andtension of tissues quickly receded. A total recession of pain followedwithin the next several hours, as was observed in the cases involvinggenital herpes. Physical examinations showed that the swelling andblisters disappeared within 2-3 days. In individual cases, it took up tofive days for the conditions to completely clear and for the entirehealing process to be accomplished. It was further observed that inpatients whose treatment started on the first day of the disease, skinconditions such as swelling, irritations, and papules disappearedtotally after 24 hours. Another observation was that persons sufferingfrequent reoccurrences of this disease had prolonged periods betweenreoccurrences and that the reoccurring symptoms were milder each time.In no cases were any side effects observed.

EXAMPLE 13

Clinical research was done on six patients with herpes zoster who weretreated with a dimer composition of the present invention. In fivecases, the disease developed in a typical manner, and the sixth case hadparticular complications which will be discussed below. These patientswere treated with an ointment containing pancreatic ribonuclease Adimer, and the treatment was begun on the third or fourth day of thedisease. Applications of the ointment were made 4-5 times daily.

In all cases, pain receded totally within the first 24-48 hours.Blisters were observed to dry up after 3-4 days, and thus after thisperiod it was decided to terminate the treatments. Within the next fewdays, the skin conditions healed completely. In none of the patients didthe pain persist after around the first 24-48 hours, and conditions didnot reappear in three patients remaining under observation for more thanone year. In one patient, as indicated above, there was an unusualclinical development of the disease. A woman aged 42 had been treatedfor lung cancer using cobalt therapy which had seriously impaired herimmunity system. Apart from the usual symptoms of herpes zoster, therewas found a generalized dissemination of blisters all over her body. Thetreatment using the ribonuclease dimer composition of the presentinvention was totally successful in this immunity-impaired patient, andthe woman showed complete recession of the blister condition after a fewdays. The successful results in this patient and in the other members ofthe group shows the particular effectiveness of pancreatic ribonucleaseA dimer against herpes zoster caused by Varicella viruses.

What is claimed is:
 1. A method of treating viral infections comprisingadministering to a human or animal subject in need of such treatment aneffective antiviral amount of a dimer of lysozyme and a pharmaceuticallyacceptable carrier.
 2. A method according to claim 1 wherein thecomposition is administered orally, intravenously or topically.
 3. Amethod according to claim 1 wherein the carrier is a physiologicallyacceptable salt solution.
 4. A method according to claim 3 wherein saidsalt solution of a 0.5-1.5% salt solution in water.
 5. A methodaccording to claim 1 wherein the effective amount of lysozyme dimer isfrom 0.1 to 5 mg/kg of body weight.
 6. A method according to claim 5wherein the effective amount is 1 to 2 mg/kg of body weight.
 7. A methodaccording to claim 1 for treating infection caused by parvovirus.
 8. Amethod according to claim 1 wherein lysozyme dimer is administered in aconcentration of 0.001 to 10 mg/ml.
 9. A method according to claim 1,for inhibiting the replication of Sendai virus.